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Description of key information

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records
Reference
Endpoint:
skin sensitisation
Remarks:
in vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
18 September 2002 to 02 October 2002
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
Study conducted in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of the relevant results. The study report was conclusive, done to a valid guidelines and the study was conducted under GLP conditions.
Qualifier:
according to guideline
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Deviations:
no
Qualifier:
according to guideline
Guideline:
OECD Guideline 406 (Skin Sensitisation)
Deviations:
no
GLP compliance:
yes
Type of study:
mouse local lymph node assay (LLNA)
Species:
mouse
Strain:
CBA
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Age at study initiation: 7 - 12 weeks (beginning of acclimation period).
- Weight at study initiation: 16.1-21.3 g (beginning of acclimation period).
- Housing: In groups of 4 (by dose), in cages with standards softwood bedding.
- Diet: Pelleted standard mouse maintenance diet, provided ad libitum.
- Water: Tap water, ad libitum.
- Acclimation period: 6 days (18 September 2002 to 24 September 2002).

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3 ºC
- Humidity (%): 30-70 %
- Air changes (per hr): 10-15 changes per hour.
- Photoperiod (hrs dark / hrs light): 12 hours dark / 12 hours light (artificial).

IN-LIFE DATES: From: 18 September 2002 To: 02 September 2002
Vehicle:
other: ethanol:water, 7:3 (v/v)
Concentration:
0.0 (vehicle control), 0.1, 1, 10 and 100%.
No. of animals per dose:
4
Details on study design:
MAIN STUDY
- Criteria used to consider a positive response: Firstly the criteria for a positive response is that one or more concentration of the test material should elicit a 3-fold or greater increase in the incorporation of ³H-methyl thymidine in comparison to the vehicle control, as indicated by the stimulation index. Secondly, that the data are compatible with a conventional dose response. Conversely, a test material which does not fulfil the above criteria is designated as unlikely to be a sensitizer.

TREATMENT PREPARATION AND ADMINISTRATION:
- Preparation of test solution: The test material was placed in a volumetric flask and the vehicle (ethanol:water, 7:3 (v/v)) was quantitatively added. The weight volume dilutions were prepared individually using a magnetic stirrer as a homogenizer. Homogeneity of the test solution was maintained during treatment with the magnetic stirrer. Preparations were made shortly before each dosing. The test solutions were analysed at four selected consecutive concentrations.
- Administration: Test solutions were topically applied to the dorsum of each ear lobe (left and right) on three consecutive days at an application volume of 25 µL. The area was dried with hot air after application, to avoid loss of the test material.
- Post application procedure: Five days after the first topical application the mice were injected intravenously into a tail vein with 250 µL of radio-labelled thymadine (³H-methyl thymidine). Approximately five hours after intravenous injection, the mice were sacrificed, the draining auricular lymph nodes excised and pooled per group. Single cell suspensions (phosphate buffered saline) lymph node cells were prepared from pooled lymph nodes which were washed and incubated with 5% trichloroacetic acid overnight. The precipitates were then resuspended in 5% trichloroacetic acid and transferred to scintillation vials with 10 mL of scintillation cocktail. The proliferative capacity of pooled lymph node cells was determined by the incorporation of ³H-methyl thymidine measured in a ß-scintilation counter. The ß-scintilation counter expressed ³H-methyl thymidine incorporation as the number of radioactive disintegrations per minute.
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
Statistics:
Mean values and standard deviations were calculated for bodyweights.
Positive control results:
The stimulation indices of 2.9, 2.6 and 7.1 were determined with the positive control at concentrations of 5, 10 and 25% in acetone:olive oil, 4:1 (v/v). Based on the criteria, the test material was found to be a non-sensitiser when tested up to 10% (w/v) in acetone:olive oil, 4:1 (v/v). Alpha-hexylcinnamaldehyde showed an alergenic potency when tested at a concentration of 25% (w/v). EC3 is the estimated concentration for a stimulation index of 3. In this study an EC3 of 11.3% (w/v) was theoretically calculated with stimulation indices of 2.6 and 7.1 at positive control concentrations of 10% and 25% (w/v).
Parameter:
SI
Remarks on result:
other: see Remark
Remarks:
The application of the test material at concentrations of 0.1, 1, 10 % (w/v) in ethanol:water, 7:3 (v/v) and 100% (undiluted) resulted in a stimulation index which was less than 3-fold at all four concentrations, values can be seen in Table 1. Consequently, the test material was designated as unlikely to be a sensitiser under the conditions of the test.
Parameter:
other: disintegrations per minute (DPM)
Remarks on result:
other: The pooled disintegrations per minute and disintegrations per minute per lymph node are presented in Table 1 in the field "any other information on results incl. tables".

Viability/Mortality

- No deaths occurred during the study.

Clinical Signs

- No test material related clinical signs were observed in all the animals of the control group and those exposed to 0.1, 1 and 10% dilutions of the test material.

- Approximately 1 hour after the first topical application, a moderate swell was observed at both sites in all mice exposed to the test material at 100%. This lasted for four days and then reduced slightly, effects persisted until termination.

Bodyweights

- Bodyweights were within the range commonly recorded for animals of this strain and age.

Table 1: Results

Test item concentration % (w/v)   Meaurement dpm Calculation Result
dpm - BGa Number of lymph nodes dpm per lymph nodeb S.I.
BG I 3
BG II 3
CG 1 2345 2342 8 293
0.1 TG 2 2278 2275 8 284 1.0
1 TG 3 2746 2743 8 343 1.2
10 TG 4 2493 2490 8 311 1.1
100* TG 5 3720 3717 8 465 1.6
Positive control concentration % (w/v)            
BG I 3
BG II 3
CG 1 4232 4229 8 529
5 TG 2 121669 12166 8 1521 2.9
10 TG 3 10977 10974 8 1372 2.6
25 TG 4 29856 29853 8 3732 7.1

* undiluted as delivered by the sponsor.

BG = Background (1mL 5% trichloroacetic acide) in duplicated

CG = Control group

TG = Test group

S.I. = Stimulation Index

a = The mean value was taken from the figures BG I and BG II

b = Since the lymph nodes of the animals of a dose group were pooled, DPM/Node was determined by dividing the measured value by the number of lymph nodes pooled.

Interpretation of results:
not sensitising
Remarks:
Migrated information Criteria used for interpretation of results: EU
Conclusions:
Under the conditions of the test the stimulation index was less than 3-fold that of the control at all test concentrations and therefore, the test material is considered to be unlikely to be a skin sensitizer.
Executive summary:

The skin sensitisation potential of the test material was determined in accordance with the standard guidelines OECD 429 and OECD 406 using the mouse Local Lymph Node Assay (LLNA). The test material was applied as 0.1, 1, 10 % w/v preparation in ethanol:water, 7:3 (v/v) and 100% (undiluted). A vehicle control was run concurrently. The positive control was shown to have the capacity to cause skin sensitisation confirming the validity of the protocol used for the study.

Under the conditions of the test, the stimulation index induced by the test material was less than 3 -fold that of the control at all test concentrations, and therefore the test material is considered to be unlikely to be a skin sensitizer.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not sensitising)
Additional information:

The key study (Wang-Fan, 2002) determined the skin sensitisation potential of the test material under GLP conditions and in accordance with the standard guidelines OECD 429 and OECD 406 using the mouse Local Lymph Node Assay (LLNA). The test material was applied as 0.1, 1, 10 % w/v preparation in ethanol:water, 7:3 (v/v) and 100% (undiluted). A vehicle control was run concurrently. The positive control was shown to have the capacity to cause skin sensitisation confirming the validity of the protocol used for the study. Under the conditions of the test, the stimulation index induced by the test material was less than 3 -fold that of the control at all test concentrations, and therefore the test material is considered to be unlikely to be a skin sensitizer. The study was assigned a reliability score of 1 in line with Klimisch (1997) and considered suitable for assessment as an accurate reflection of the test material.

Hopf (1968) has been provided as supporting data, in which the skin sensitisation potential of the test material was determined in an open epicutaneous test. A concentrated form of test material was rubbed onto the shaved flank of 20 guinea pigs twice daily for 21 consecutive days, the induction exposure. After a 5 day break, the challenge exposure was performed on the neck of each animal, this time rubbing was performed once daily for 5 additional days. Dermal reactions were monitored throughout the experiment. Throughout the induction and challenge exposure periods, no skin reactions were noted to either the flank or the neck of the animals. During the course of the study no allergenic properties were detected. The test material was considered to be unlikely to be a skin sensitizer. The test was conducted to sound scientific principles, however reporting was insufficient. Details, crucial to the evaluation of the results, were omitted from the report, such as the test concentration used. It is therefore not possible to assess the reliability of the test results in line with Klimisch (1997), thus the study was assigned a reliability score of 4.


Migrated from Short description of key information:
Not sensitisaing, mouse LLNA, OECD 429 & 406, Wang-Fan (2002).

Justification for selection of skin sensitisation endpoint:
The key study was conducted to under GLP and the standardised guidelines OECD 429 and 406 with a sufficient level of reporting to assess the quality of the data submitted. Accordingly the study was assigned a reliability score of 1 according to the criteria of assessing data quality as defined by Klimisch (1997).

Respiratory sensitisation

Endpoint conclusion
Endpoint conclusion:
no study available

Justification for classification or non-classification

According to the criteria outlined in Regulation (EC) No. 1272/2008 and Directive 67/548/EEC, the substance does not meet the criteria for classification as a skin sensitizer.

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